Brief preconditioning ischemia results in tolerance to subsequent ischemic brain injury. The genomic signature of the tolerant brain is transcriptional suppression. The translational elements of this protein synthesis-dependent phenomenon of tolerance have been unknown. However, we now show the tolerance proteome (Stapels et al. Science Signaling, Mar 2010). The proteome is enriched in histone proteins and remarkably in Polycomb group (PcG) proteins whose molecular functions are those of transcriptional suppression. Thus a mechanism of induction of transcriptional suppression, characterizing tolerance, may have been discovered. This discovery implicates epigenetic gene repressor proteins (PcGs) as responsible for the process of transcription suppression resulting in ischemic tolerance. Further, we show that PcGs, previously known as regulators of segmentation during development in drosophila, have a novel neuroprotective function in brain. Our initial studies in ischemic-tolerance in vivo and in vitro show that the development of ischemic tolerance is dependent upon the expression of PcG proteins: knock down ablates tolerance, and over-expression produces tolerance. Further, we now have preliminary evidence in ischemic tolerance for opposing changes of Trithorax group (TrxG) proteins, the gene activator and antagonist of PcG proteins. Accordingly we will investigate PcG/TrxG epigenetic regulation to determine their effector properties upon the tolerance mechanism. We offer the following aims: Specific Aim 1. To establish in absolute molar numbers, the stoichiometry of proteins that comprise PcG/TrxG epigenetic regulator complexes at the onset of ischemic injury and the onset of ischemic tolerance. Quantitative MS analyses will be performed on brain proteins immunoprecipitated with specific antibodies against selected PcG or TrxG proteins. Specific Aim 2. To establish the identity of newly synthesized proteins in the brain at the onset of ischemic injury and the onset of ischemic tolerance. We will employ the Click technique to metabolically label the newly synthesized protein, isolate them, and quantify them with quantitative MS analyses. Bioinformatics of the nascent proteomes will be established with the assistance of bioinformatic tools.